Prostate cancer is, after skin cancer, the most common malignancy in man. There are a variety of treatment options for localized prostate cancer, but the disease becomes virtually incurable once the cancer has metastasized to the lymph nodes or distant organs. The long-term objective of this proposed research project is to elucidate the role of blood clot in the prostate tumor stroma and utilize this knowledge to treat metastatic prostate cancer. The presence of blood clot in the tumor stroma has been recognized for many years. Clotted plasma is a specific component of the tumor stroma and, therefore an excellent target for drug delivery. We recently identified two peptides CLT1 and CLT2, which upon injection into mice associate with clotted plasma proteins in the stroma of a variety of tumors including prostate cancer xenografts. However, the CLT peptides do not bind to healthy organs. CLT1 has an additional function as it is cytotoxic for proliferating endothelial and prostate tumor cells;CLT1 has no effect on confluent (non-proliferative) endothelial cells. Overall, these observations support the hypothesis that the CLT peptides bind to the prostate tumor stroma based on their affinity for a unique binding site in blood clot and that this affinity can be used to treat end stage prostate cancer. The specific aims are: (1) To isolate a protein or protein fragment from clotted plasma encompassing the CLT binding site in prostate tumor interstitial spaces. We will identify the tumor- specific clot protein (= CLT-binding protein or CLT-BP) that binds to the CLT peptides and develop an antibody to analyze the expression of the CLT-BP in clinical prostate cancer. We will also identify the minimal amino acid sequence within the CLT peptide (CLT-motif) that mediates binding to the CLT-BP. The identification of the CLT-motif will allow us to identify physiological binding partners of the CLT-BP by screening data bases for homologies. (2) To determine if CLT1 is effective in treating prostate cancer and prostate cancer metastases. We will determine whether the cytotoxicity of CLT1 translates into anti-tumor and anti-metastatic activity for prostate cancer in a mouse xenograft model in vivo and elucidate the cytotoxic function of CLT1 by analyzing its structure activity relationship in vitro. (3) To determine if androgen deprivation therapy increases homing of the D-K6L9-CLT1 fusion peptide to prostate tumors and metastases and if homing of D-K6L9-CLT1 improves the anti-tumor and anti-metastatic effect of the androgen deprivation therapy. We will determine if D-K6L9 becomes a more effective anti-tumor drug after conjugation to CLT1 which is expected to direct D-K6L9 to clot in prostate tumor necroses caused by androgen ablation. The results of this study will lead to novel information about the structure and function of the prostate tumor stroma. This knowledge will be immediately translated into new treatment options for metastatic prostate cancer. PUBLIC HEALTH RELEVANCE. Prostate cancer is, after skin cancer, the most common malignancy in male Americans and becomes virtually incurable once the cancer has spread to the lymph nodes or distant organs. This project will determine if treatment with the CLT peptides can provide a cure or at least a therapy that inhibits prostate cancer from further spreading without causing major side effects. Delineating the interaction of CLT1 and CLT2 with the prostate tumor is an important milestone for further developing the peptides into a useful drug or drug carrier.